Analysis of GzmbCre as a Model System for Gene Deletion in the Natural Killer Cell Lineage.

Xu Y, Evaristo C, Alegre ML, Gurbuxani S, Kee BL - PLoS ONE (2015)

Bottom Line:
We demonstrated the utility of this model by creating GzmbCre;Rosa26IKKbca mice in which Cre-mediated recombination resulted in expression of constitutively active IKKβ, which results in activation of the NFκB transcription factor.As a caveat to the use of GzmbCre we found that this transgene can lead to recombination in all hematopoietic cells the extent of which varies with the particular loxp flanked allele under investigation.We conclude that GzmbCre can be used under some conditions to investigate gene function in mature and activated natural killer cells.

Affiliation: Committee on Molecular Pathogenesis and Molecular Medicine, University of Chicago, Chicago, Illinois, United States of America.

ABSTRACTThe analysis of gene function in mature and activated natural killer cells has been hampered by the lack of model systems for Cre-mediated recombination in these cells. Here we have investigated the utility of GzmbCre for recombination of loxp sequences in these cells predicated on the observation that Gzmb mRNA is highly expressed in mature and activated natural killer cells. Using two different reporter strains we determined that gene function could be investigated in mature natural killer cells after GzmbCre mediated recombination in vitro in conditions that lead to natural killer cell activation such as in the cytokine combination of interleukin 2 and interleukin 12. We demonstrated the utility of this model by creating GzmbCre;Rosa26IKKbca mice in which Cre-mediated recombination resulted in expression of constitutively active IKKβ, which results in activation of the NFκB transcription factor. In vivo and in vitro activation of IKKβ in natural killer cells revealed that constitutive activation of this pathway leads to natural killer cell hyper-activation and altered morphology. As a caveat to the use of GzmbCre we found that this transgene can lead to recombination in all hematopoietic cells the extent of which varies with the particular loxp flanked allele under investigation. We conclude that GzmbCre can be used under some conditions to investigate gene function in mature and activated natural killer cells.

pone.0125211.g003: GzmbCre;Rosa26ACTB-tdTomato, -EGFP/+ mice have a low frequency of GFP+ mature NK cells.(A) Representative FACS histograms showing the gating strategy for mNK cells in GzmbCre;Rosa26ACTB-tdTomato, -EGFP/+ mice. Lymphoid cells were gated by FSC and SSC (left panel) and then Lineage negative, PI- cells (center plot) were selected and examined for expression of NK1.1 and CD122. (B) The frequency of mNK cells expressing GFP in a typical GzmbCre;Rosa26ACTB-tdTomato, -EGFP/+ mouse bone marrow (BM), spleen, lymph node (LN) and liver. The frequency of GFP+ cells is indicated. (C) Scatter plots showing the frequency of YFP+ mNK cells in the bone marrow of multiple GzmbCre;Rosa26ACTB-tdTomato, -EGFP/+ mice at different ages. Each circle represents one mouse. (D) The average number of GFP+ mNK cells is shown for each of the indicated tissues in mice between 6 and 12 weeks of age. Error bars = standard deviation. n > 7 for each tissue. The difference in average GFP+ mNK cell number between different tissues was not statistically significant.

Mentions:
(A) Representative FACS histograms showing the gating strategy for mNK cells in GzmbCre;Rosa26EYFP mice. Lymphoid cells were gated by FSC and SSC (left panel) and then Lineage/PI negative cells that express CD122 (center plot) were selected and examined for expression of NKp46 and DX5. In some experiments mNK cells were Lineage/PI negative CD122+NK1.1+ as shown in Fig 3A. (B) The frequency of mNK cells expressing EYFP in a typical GzmbCre;Rosa26EYFP mouse bone marrow (BM), spleen (spl), lymph node (LN) and liver. (C) Scatter plots showing frequency of YFP+ mNK cells in the bone marrow of multiple GzmbCre;Rosa26EYFP mice at different ages. Each circle represents one mouse. (D) The average number of YFP+ mNK cells is shown for each of the indicated tissues in mice between 6 and 12 weeks of age. Error bars = standard deviation. n > 5 for each tissue. The difference in average YFP+ mNK cell number between different tissues was not statistically significant.

pone.0125211.g003: GzmbCre;Rosa26ACTB-tdTomato, -EGFP/+ mice have a low frequency of GFP+ mature NK cells.(A) Representative FACS histograms showing the gating strategy for mNK cells in GzmbCre;Rosa26ACTB-tdTomato, -EGFP/+ mice. Lymphoid cells were gated by FSC and SSC (left panel) and then Lineage negative, PI- cells (center plot) were selected and examined for expression of NK1.1 and CD122. (B) The frequency of mNK cells expressing GFP in a typical GzmbCre;Rosa26ACTB-tdTomato, -EGFP/+ mouse bone marrow (BM), spleen, lymph node (LN) and liver. The frequency of GFP+ cells is indicated. (C) Scatter plots showing the frequency of YFP+ mNK cells in the bone marrow of multiple GzmbCre;Rosa26ACTB-tdTomato, -EGFP/+ mice at different ages. Each circle represents one mouse. (D) The average number of GFP+ mNK cells is shown for each of the indicated tissues in mice between 6 and 12 weeks of age. Error bars = standard deviation. n > 7 for each tissue. The difference in average GFP+ mNK cell number between different tissues was not statistically significant.

Mentions:
(A) Representative FACS histograms showing the gating strategy for mNK cells in GzmbCre;Rosa26EYFP mice. Lymphoid cells were gated by FSC and SSC (left panel) and then Lineage/PI negative cells that express CD122 (center plot) were selected and examined for expression of NKp46 and DX5. In some experiments mNK cells were Lineage/PI negative CD122+NK1.1+ as shown in Fig 3A. (B) The frequency of mNK cells expressing EYFP in a typical GzmbCre;Rosa26EYFP mouse bone marrow (BM), spleen (spl), lymph node (LN) and liver. (C) Scatter plots showing frequency of YFP+ mNK cells in the bone marrow of multiple GzmbCre;Rosa26EYFP mice at different ages. Each circle represents one mouse. (D) The average number of YFP+ mNK cells is shown for each of the indicated tissues in mice between 6 and 12 weeks of age. Error bars = standard deviation. n > 5 for each tissue. The difference in average YFP+ mNK cell number between different tissues was not statistically significant.

Bottom Line:
We demonstrated the utility of this model by creating GzmbCre;Rosa26IKKbca mice in which Cre-mediated recombination resulted in expression of constitutively active IKKβ, which results in activation of the NFκB transcription factor.As a caveat to the use of GzmbCre we found that this transgene can lead to recombination in all hematopoietic cells the extent of which varies with the particular loxp flanked allele under investigation.We conclude that GzmbCre can be used under some conditions to investigate gene function in mature and activated natural killer cells.

Affiliation:
Committee on Molecular Pathogenesis and Molecular Medicine, University of Chicago, Chicago, Illinois, United States of America.

ABSTRACTThe analysis of gene function in mature and activated natural killer cells has been hampered by the lack of model systems for Cre-mediated recombination in these cells. Here we have investigated the utility of GzmbCre for recombination of loxp sequences in these cells predicated on the observation that Gzmb mRNA is highly expressed in mature and activated natural killer cells. Using two different reporter strains we determined that gene function could be investigated in mature natural killer cells after GzmbCre mediated recombination in vitro in conditions that lead to natural killer cell activation such as in the cytokine combination of interleukin 2 and interleukin 12. We demonstrated the utility of this model by creating GzmbCre;Rosa26IKKbca mice in which Cre-mediated recombination resulted in expression of constitutively active IKKβ, which results in activation of the NFκB transcription factor. In vivo and in vitro activation of IKKβ in natural killer cells revealed that constitutive activation of this pathway leads to natural killer cell hyper-activation and altered morphology. As a caveat to the use of GzmbCre we found that this transgene can lead to recombination in all hematopoietic cells the extent of which varies with the particular loxp flanked allele under investigation. We conclude that GzmbCre can be used under some conditions to investigate gene function in mature and activated natural killer cells.